Construction And Application Of Novel Biosensors Based On Nucleic Acid Amplification Technology And Nanomaterials

Nucleic acids,small biomolecules and proteins are involved in many important life processes,and their detection has received more and more attention.Because of their high sensitivity,high specificity and high accuracy,optical biosensors are widely used in the detection of biomolecules.Nanomaterials have excellent optical,electrical,magnetic and catalytic properties due to their small size,variable structure,large specific surface area and other characteristics,making nanomaterials have great advantages in the microelectronics industry,catalysis,analysis and detection,life medicine and other fields,and have been widely studied and applied.Nanomaterials provide new tools for analytical chemistry.The introduction of appropriate signal amplification mechanism and the further construction of molecular recognition elements are expected to break through the shortcomings and limitations of traditional biosensor detection,thus providing new tools and new opportunities for life analysis.In this paper,combining the advantages of nanomaterials and nucleic acid amplification technology,a series of sensing methods are established for the detection of biomolecules,including the following three aspects:1.A fluorescent sensor based on polydopamine nanotubes(PDANTs)was constructed for the highly sensitive detection of T4 polynucleotide kinase phosphatase(T4 PNKP).The PDANTs have strong adsorption for single-stranded DNA(ssDNA),but have week adsorption for double-stranded DNA(dsDNA).In the absence of T4 PNKP,the FAM-labelled ssDNA was absorbed on the surface of PDANTs.Due to the fluorescence resonance energy transfer between the FAM-labelled ssDNA and PDANTs,the fluorescence of ssDNA was quenched.In the presence of T4 PNKP,the 3'-phosphate terminal of ssDNA is catalyzed to form hydroxyl terminal.With the addition of dNTPs and KF polymerase,3'-hydroxyl terminal is extended to form dsDNA.Because of the week affinity between PDANTs and dsDNA,the dsDNA could retain most of the fluorescence.The proposed method demonstrates the sensitivity for T4 PNKP assay in the range from 0.05 to 1.5 U mL-1 with the detection limit of 0.005 U mL-1.The proposed fluorescence method may provide a new platform for drug screening and disease-related research.2.A novel fluorescent methd based on hybridization chain reaction(HCR)induced MoS2 quantum dots inner filter effect was proposed for the highly sensitive detection of microRNA.When the target microRNA was added to the system,the microRNA and the hairpin DNA probe HP1 were hybridized to induce the HCR cycle,and the H1 and H2 were assembled into DNA nanowires containing G-quadruplex.Hemin/Gquadruplex DNAzyme could be formed when hemin is inserted into G-quadruplex.With the assistance of H2O2,hemin/G-quadruplex DNAzyme could oxidize ophenylenediamine(OPD)to 2,3-diaminophenazine(DAP).Due to the inner filter effect between DAP and MoS2 quantum dots,the fluorescence of MoS2 quantum dots is quenched.The detection limit of this method is 42 fM,and the detection range is 0100 pM,which provides a platform for the detection of microRNA in biomedical research and clinical analysis.3.A highly sensitive and specific methd based on catalyzed hairpin assembly(CHA)and hybridization chain reaction(HCR)induced WS2 quantum dots inner filter effect was developed for the detection of microRNA.When the target microRNA is added to the system,the hairpin DNA probe HP recognize the microRNA and combined the DNA probes H1 and H2 to trigger the CHA cycle.The hybridization of the CHA product with the DNA probe H3 triggers the HCR cycle,which makes the DNA probes H3 and H4 self-assemble into DNA nanowires containing G-quadruplex.G-quadruplex and hemin combine to form hemin/G-quadruplex DNAzyme.In the presence of H2O2,hemin/G-quadruplex DNAzyme oxidizes o-phenylenediamine(OPD)to 2,3diaminophenazine(DAP).Due to the inner filter effect between DAP and WS2 quantum dots,the fluorescence of WS2 quantum dots is quenched.This method has high sensitivity and good selectivity with a detection limit of 2.7 fM.The effective cascade amplification of this method provides a powerful platform for the detection of microRNAs in serum.